Method for obtaining beta casein

ABSTRACT

PCT No. PCT/FR91/00506 Sec. 371 Date Nov. 13, 1992 Sec. 102(e) Date Nov. 13, 1992 PCT Filed Jun. 25, 1991 PCT Pub. No. WO92/00017 PCT Pub. Date Jan. 9, 1992.A method for obtaining beta casein by separating it from para kappa casein involving the use of rennet casein is disclosed.

The invention relates to the obtaining of beta casein from rennetcasein. It relates more particularly to a method and also to a deviceadapted to obtain a solution of beta casein from a suspension orsolution of rennet casein.

It is known to obtain rennet casein from the precipitation of micells ofcaseins of a mammal milk after hydrolysis of the casein by the rennet orby any other enzyme or mixture of enzymes of animal, vegetable,bacterial or fungal origin capable of hydrolyzing the pHe-Met 105-106bond of kappa casein. After the separation of the lactoserum and thewashing of the curds, an insoluble product is obtained which containsall the caseins of the milk thus used, except for the hydrophilicfraction which stabilizes the micell and is released by theenzyme--i.e., the caseino macropeptide. The other product of theenzymatic reaction is referred to as para kappa casein.

Amongst the caseins present in this insoluble product produced by thecurdling of mammal milks, beta casein is known to have numerousfunctional, technological and physiological properties bound up with itsphysico-chemical characteristics. These properties include moreparticularly:

foaming and emulsifying properties conferred on beta casein by itsstrong hydrophobicity. This property also plays a part in thestabilization of the micellar structure in association with thecolloidal phosphate and plays a part in the texture of cheese-makingcurds;

nutritional properties resulting from its amino acid composition,providing considerable richness in lysine and tryptophane;

properties enabling it to be used in the pharmaceutical industry, sincethe hydrolysis of beta casein leads to:

the obtaining of phosphopeptides which play a role in the intestinalabsorption of mineral elements, and

the formation of a hexapeptide known as beta casomorphine, which seemsto play a role comparable to opiate derivatives which influence sleep,the secretion of insulin and control of the appetite.

It is known that beta casein has the property of becoming solubilizedwhen the temperature goes down and the quantity of beta caseindissociating cold from the micell increases in the course of time,although 75% of this quantity is released in the first 15 minutes(CREAMER et al, NZ J. Dairy Science Technol. 12, 58-66, 1977).

The quantity of beta casein capable of cold solubilization also dependson the protein concentration used and also on the concentration ofcolloidal phosphate present (PIERRE and BRULE, Journal of Dairy Research48, 417-428, 1981).

Having regard to the numerous properties of beta casein, it is clearlyimportant to develop a process which can be economically used inindustry and which enables beta casein to be separated from the othercaseins present in the medium. In the past a certain number of attemptsto solve this problem have been made.

One example of such an attempt is represented by French Patent No. 2 592769, which discloses a method of obtaining a substance enriched in betacasein by separation of the beta casein from a mammal milk or aderivative of mammal milk, such as a caseinate, the process being moreparticularly performed by a molecular sieving technique using amicro-filtration membrane.

This prior art technique has a certain number of disadvantages, amongstwhich the following may be noted:

a difficulty in obtaining at one and the same time a high beta caseinconcentration and adequate purity, and

extremely low cold micro-filtration performances, something which formsa certain handicap to the extrapolation of this process to theindustrial level.

It is therefore an object of the invention to provide a new method forobtaining beta casein which is free from the disadvantages of the priorart methods and which can be used industrially. The invention startsfrom the realization that beta casein can be extracted from rennetcasein and that such extraction gives excellent yields both inconcentration and purity.

Accordingly a first aspect of the invention has for its object a processfor obtaining a solution of beta casein from rennet casein as producedby the enzymatic curdling of milk. One of the original features of theprocess according to the invention in comparison with the prior arttechnique disclosed in French Patent No. 2 592 769 resides in the usenot of a caseinate, but of a rennet casein in which kappa casein, themain contaminant found in the extraction of beta casein, is hydrolyzedto form para kappa casein, which has a much lower solubility.

The method according to the invention is therefore characterized in thatthe suspension or solution of rennet casein, for example, as formed bythe enzymatic curdling of a mammal milk is cooled to a temperature ofthe order of approximately -2° C. to +10° C., preferably between +2° C.and +5° C. and its pH is adjusted to a value of approximately 4.00 to5.00, and separating the suspension or solution of rennet casein thuscooled and acidified into two phases, so as to obtain a solid phase anda liquid phase, the latter containing the beta casein.

In the method according to the invention it is possible to use moreparticularly as the mammal milk, cows or goats milk, the rennet caseinoriginating from the enzymatic curdling of said mammal milk by a mixtureof enzymes known as rennet enzymes or by an enzyme of animal, vegetable,bacterial, fungal origin or by a mixture of these different enzymesenabling the casein to be hydrolyzed.

The rennet casein can be used in suspension in water or in a salinesolution and can thus be partially or completely solubilized. The saltswhich can be used include more particularly sodium, potassium orammonium chlorides, sodium, potassium or ammonium citrates, sodium,potassium or ammonium oxalates, sodium, potassium or ammoniumphosphates, the salts being usable on their own or in various mixtures.Preferably the concentration of salts or of a mixture of different saltsused in the solution can vary from approximately 0.1 to 4%.

Preferably according to the invention the concentration of rennet caseinpresent in the suspension or solution can vary from 1 to 10%, preferablyfrom approximately 4 to 7%.

In the first stage of the method according to the invention the pH ofthe cooled suspension or solution of rennet casein is adjusted to theaforementioned value of approximately 4.00 to 5.00 by the addition of anorganic or mineral acid, or of a mixture of these two types of acids.The organic acid used can be more particularly acetic acid, citric acid,lactic acid, oxalic acid, either separately or in various mixtures. Themineral acid which can be used is more particularly hydrochloric acid,sulphuric acid, nitric acid, phosphoric acid, either separately or invarious mixtures. Of course, the pH of the suspension or solution ofrennet casein is so controlled as to be kept constant during the wholeof this first phase of the method according to the invention.

According to the invention the step of acidification and control of thepH of the solution or suspension of rennet casein can be performed priorto the stage of adjusting the temperature to values required by themethod and set forth hereinbefore.

Following this chemical step of the method, the suspension or solutionof rennet casein which has thus been cooled and whose pH has beenadjusted is present in the form of a mixture of two phases:

a liquid phase containing the beta casein and

a sedimentable phase containing the remainder of the unsolubilizedcaseins.

In the second step of the method according to the invention, the twophases are separated physically either by natural decantation, or usinga suitable apparatus, as will be disclosed hereinafter in thedescription of an embodiment of the device for the performance of themethod according to the invention.

After said separation the liquid phase enriched in beta casein isrecovered by any suitable means, as will be disclosed hereinafter.

The method defined hereinbefore makes beta casein available which has adegree of purity higher than approximately 90% in relation to the totalprotein material, as measured by HPLC and electrophoresis. This productis advantageously free from chemical additives such as those used in theprior art separating techniques, such as denaturing agents,precipitating agents or urea. It is a novel product which enters as suchinto the scope of the invention.

This product is more particularly characterized in that it containspractically no kappa casein.

The beta casein as obtained by the method according to the invention isalso characterized in that it has a pH close to neutrality. Theproperties of this product are advantageously used as a food or foodcomplement in the foodstuffs and dietetic, cheese-making and dairyindustries. In dietetics it forms a very advantageous raw material forobtaining peptides.

The beta casein according to the invention is also very advantageous inthe pharmaceutical or cosmetics industry.

The invention also relates to the coproduct of the beta casein isolatedaccording to the invention, represented by the solid phase formed duringthe cooling and acidification of the suspension or solution of rennetcasein. This coproduct, which is substantially totally free from betacasein, has original functional, physiological and nutritionalproperties.

It therefore finds applications in the aforementioned industries. Thiscoproduct can also be used as a raw material for the purification of theother contaminants of micells, such purification being facilitated bythe fact that this coproduct contains practically no beta casein.

In a second aspect, the invention relates to a device for theperformance of the method as defined hereinbefore, the device beingcharacterized in that it comprises:

a reactor into which the solution or suspension of rennet caseinproduced by the enzymatic curdling of mammal milk is introduced;

cooling means enabling the temperature of the suspension in the reactorto be maintained at a value of approximately -2° C. to +10° C.,preferably between +2° C. and 5° C.;

a pH-stat enabling the quantity of acids added to the solution orsuspension of rennet casein contained in the reactor to be regulated,under the control of a pH-meter, and

means for ensuring the separation of the liquid and sedimentable phasescontained in the mixture originating from said reactor.

Other features and advantages of the invention will be gathered from thefollowing description, with reference to the accompanying drawings whichillustrate various completely non-limiting embodiments thereof andwherein:

FIG. 1 is a diagram illustrating the steps of the method according tothe invention for obtaining the solution of beta casein;

FIG. 2 shows diagrammatically the device for the performance of themethod;

FIGS. 3 to 5 are diagrams illustrating the various embodiments of themeans for separating the two phases coming from the first step of themethod according to the invention; and

FIG. 6 shows the chromatographic profile of a sample (HPLC).

Referring to the drawings, the solution or suspension of rennet casein Cis introduced into a jacketed receptacle or reactor 10, so as tomaintain therein the temperature of said solution or suspension at thevalue specified by the method--i.e., between approximately -2° C. and+10° C., preferably between +2° C. and +5° C. A cooling solutioncirculates in the jacket of the reactor 10 and the solution orsuspension of rennet casein C is subjected to slight agitation by meansof a variable speed agitator 12. The temperature is controlled, forexample, by a thermometer 14.

The pH of the cooled solution or suspension of beta casein C ismaintained at the constant value specified by the method mentionedhereinbefore by means of a pH-stat 16, which allows the control of thequantity of acid delivered by a metering pump 20 from a feed 18 tankunder the control of a pH-meter 22. The pH-stat 16 is given a requiredvalue and the metering pump 20 enables the pH of the solution orsuspension C to be adjusted in accordance with said required value, bydelivering to the reactor 10 the necessary quantity of acid (or mixtureof acids).

As indicated hereinbefore in the description of the method according tothe invention, the suspension or solution coming from the reactor 10takes the form of a mixture of two phases: a liquid phase and asedimentable phase, and the device comprises means enabling the twophases to be separated so as to enable the liquid phase containing thebeta casein to be recovered.

According to the invention the mixture of the two phases coming from thereactor 10 can be vehicled either directly by gravity (arrow 24 in FIG.2) or by means of a volumetric pump 26 or centrifugal pump 28, via aconduit which may be cooled or not.

The means used to separate the liquid phase containing the beta caseinfrom the sedimentable phase can be formed, for example, by:

a clarifier of self-clearing or non-self-clearing type operating with anacceleration from approximately 500 to 8000 g, preferably from 1500 to3000 g;

a decanter operating with an acceleration of approximately 500 to 8000g, preferably 1500 to 3000 g, or

a centrifuge operating with an acceleration of 500 to 8000 g.

These means can be cooled or not, in dependence on the varying dwelltime of the products in the installation.

Outstanding results can also be obtained in the separation of the twophases by performing decantation at atmospheric pressure into thereceptacle containing the mixture of the two phases.

FIG. 3 illustrates this embodiment. A receptacle 30 receives the mixtureof the two phases coming from the reactor 10. In the receptacle theliquid phase, which represents the supernatant enriched with betacasein, is recovered either via an orifice 32, with which the wall ofthe receptacle 30 is formed for this purpose immediately above theprecipitated phase (sediments 34), or by simple siphoning, possiblyusing a volumetric pump or a centrifugal pump (this possibility isillustrated by arrow 36 in FIG. 3).

The diagram in FIG. 4 shows a centrifuge or clarifier 38 enabling thetwo phases of the solution C coming from the reactor 10 to be separated.In this variant the liquid phase containing the beta casein isrecovered, for example by siphoning (indicated diagrammatically by arrow40), and the sediments are evacuated via a conduit 42.

Lastly, the diagram in FIG. 5 illustrates the variant using a decanter44 for the separation of the two phases. The liquid phase containing thebeta casein is recovered at place 46 and the sediment evacuated via aconduit 48.

The device according to the invention disclosed hereinbefore can operateeither intermittently or continuously.

The following is a Table showing the results of test performances of themethod according to the invention. The Table includes five embodimentsof the invention for obtaining a solution of beta casein from rennetcasein with different concentrations and using different continuously orintermittently operating devices. Of course, these examples merelyillustrate the method according to the invention and are in no waylimiting. The same thing applies to the device disclosed hereinbeforewith reference to the accompanying drawings, of which variantembodiments may be envisaged without exceeding the scope of theinvention.

                  TABLE                                                           ______________________________________                                                Separ-  Flow                                                          Raw     ator    rate    Force  Supernat-                                                                            C    C                                  material                                                                              type    m3/h    g      ant %  g/l  purity                             ______________________________________                                        1   Rennet  C.n.c.        2500   93     1.3  >99                                  casein                                                                    2   Rennet  D c     1            90     3.5  95                                   casein                                                                    3   Rennet  C.n.c.        2500   90     3.8  95                                   casein                                                                    4   Rennet  C.n.c.        2500   86     5.3  95                                   casein                                                                    5   Rennet  DG c    1            86     5.0  >98                                  casein                                                                    ______________________________________                                         n.c = intermittent operation                                                  c = continuous operation                                                      C = decanter                                                                  DG = Guinard decanter                                                    

FIG. 6 shows a chromatographic profile (HPLC reverse phase analysis) ofa sample corresponding to the following analysis:

    ______________________________________                                        Total solids   995    g/kg                                                    Nitrogen       945    g/kg beta casein > 900 g/kg                             Mineral substances                                                                           50     g/kg                                                    Calcium        5      g/kg                                                    Sodium         16     g/kg                                                    Potassium      0.1    g/kg                                                    Phosphoruses   11.1   g/kg                                                    ______________________________________                                    

The values indicated in FIG. 6 corresponds to the elution time. Theobtaining of a peak at 12.65 proves that the product obtained has apurity greater than 90%.

We claim:
 1. A method for obtaining beta casein comprising the steps:ofa) enzymatically curdling mammalian milk, to form a suspension orsolution of rennet casein comprising para kappa casein and beta casein,b) cooling the thus produced suspension or solution of rennet casein, c)adjusting the pH to acid values, whereby said cooling and pH adjustmentof said solution or suspension of rennet casein causes the formation ofa solid and a liquid phase, d) separating said solid phase from saidliquid phase, the former containing the para kappa casein and the lattercontaining the beta casein.
 2. The method according to claim 1 whereinthe enzyme used is selected from the group consisting of a mixture ofenzymes of rennet type, an enzyme of animal, vegetable, bacterial orfungal origin, or a mixture of said different enzymes.
 3. The methodaccording to claim 1, wherein the cooling step is carried out at atemperature of +2° C. to +5° C.
 4. The method according to claim 1wherein the concentration of the rennet casein present in the suspensionor solution of rennet casein thus produced is of the order of 1 to 10%.5. The method according to claim 1 wherein the rennet casein is dilutedin water or in a saline solution formed by water and 0.1 to 4% of saltsselected from sodium, potassium or ammonium chlorides, sodium, potassiumor ammonium citrates, sodium, potassium or ammonium oxalates, sodium,potassium or ammonium phosphates or mixtures thereof prior to cooling orpH adjustment.
 6. The method according to claim 1 wherein the pH of thecooled solution is adjusted by the addition of an organic acid or amineral acid or a mixture of said two types of acid.
 7. The methodaccording to claim 6, wherein the organic acid used is acetic acid,citric acid, lactic acid, oxalic acid, or mixtures thereof, the mineralacid used being hydrochloric acid, sulfuric acid, phosphoric acid,nitric acid, or mixtures thereof.
 8. The method according to claim 1wherein the step of acidification of the solution or suspension ofrennet casein is performed prior to the stage of controlling thetemperature of said solution or suspension to values ranging from -2° C.to +10° C.
 9. The method according to claim 4, wherein the concentrationof rennet casein is of 4 to 7%.
 10. The method according to claim 1,wherein said separation is performed by decantation at atmosphericpressure.
 11. The method according to claim 1, wherein said separationis performed by clarification decantation.
 12. The method according toclaim 1, wherein said separation is performed by centrifugation.
 13. Amethod for obtaining beta casein comprising the steps ofa) enzymaticallycurdling mammalian milk, to form a suspension or solution of rennetcasein comprising para kappa casein and beta casein, b) cooling the thusproduced suspension or solution of rennet casein to a temperature ofbetween -2° C. and +10° C., c) adjusting the pH to a value of 4.00 to5.00, d) separating the thus cooled and acidified suspension or solutionof rennet casein into a solid phase and a liquid phase, the formercontaining the para kappa casein, the latter containing the beta casein.